FLUORESCENT SENSORS FOR SUBCELLULAR COPPER IMAGING IN LIVE-CELLS Project Summary Background and significance: Copper is a trace metal nutrient essential for most forms of life and is the third most abundant transition metal in humans. The redox activity of copper is critical for several key physiological processes; however, unregulated levels of copper can induce oxidative stress and toxicity in cells. Either copper deficiency or copper over-load in the body is linked to various disease conditions. The biochemical basis for the connection between copper imbalance and human disease remains obscure. Measurement of copper ions in cellular or subcellular compartments is a challenging task due to the lack of analytical techniques with adequate spatial and temporal resolution and sensitivity. To fill this gap, this research project focuses on the development of new ?turn-on? and ratiometric fluorescent sensors designed to track the accumulation, speciation and trafficking of copper ion in live cells by optical imaging, which will facilitate detailed studies of the role of this ion in human health and disease. Coordination-induced fluorescence resonance energy transfer (CIFRET) mechanisms will be applied to develop the copper-imaging probes. The probes will be capable of entering subcellular compartments such as mitochondria to image copper at subcellular level and their ability to image copper ions will be tested in live cells. Fluorescent probes have become indispensable tools in modern biomedicine and biotechnology because they provide real time information concerning the quantity of ions or molecules of interest within the living cell. Thus, the copper-specific fluorescent sensors developed in this project may spur significant advances in our understanding of cell biology. Most of the reported probes undergo fluorescence quenching upon binding with copper ion and the fluorescence changes can only be observed in non-aqueous solvent, which greatly limits their potential biological application. Therefore, developing new ?turn-on? and ratiometric fluorescent probes for copper ion that behave well under physiological conditions is highly desirable. An integral part of this project is the use of density functional theory (DFT) and time-dependent DFT to understand the ability of copper ion binding, energy stabilization and fluorescence imaging. The goal of this project will be accomplished by the following specific aims. Aim 1: Develop an efficient and environmentally friendly microwave assisted organic synthesis for the development of probes to detect copper ion in human neuroblastoma SH-SY5Y cells. Our efforts will be focused on the development of rhodamine-coumarin derivatives with good water solubility, cell permeability and photostability. In the development of sensors, care will be taken to utilize and development in environmentally friendly process. Aim 2: Develop sensors effective in aqueous media and chemical tools for the study of biological copper by fluorescence-based techniques. The strategy involving modifies the structure of the receptor with water soluble groups that enhance the sensing activity of biological copper in aqueous and living cells. The role of various factors affecting the analytical characteristics of fluorescent sensors, their selectivity and sensitivity toward biological copper, as well as other important characteristics will be studied. Aim 3: Examine the cytotoxicity of rhodamine-coumarin probes and fluorescence imaging of copper ion in human neuroblastoma SH-SY5Y cells. Owing to biocompatibility nature of rhodamine-coumarin dyes, the ability of the probes for detection and localize of copper in neuroblastoma SH-SY5Y cells will be examined by fluorescence microscope imaging. Furthermore, this proposal will enhance the infrastructure of research and education at Morgan State University, introducing biochemical and biomedical research experience to underrepresented minority and female students, who would otherwise lack such opportunities. This would allow them to experience a broad spectrum of techniques, and acquire skills such as data analysis used in modern scientific investigations, while developing a vast network of partnership among scientists from national and international institutes.